Generally, 5-aminolevulinic acid (ALA) is a major precursor of tetrapyrrole compounds such as heme, bacteriochlorophylls, corrinoid and the like in all organisms, and is a photodynamic compound that forms Pchlide, a strong oxidant, by sunlight. The Pchlide induces a series of oxidation reactions that selectively destroy phospholipids in the leaves of dicotyledonous plants to kill the plants. Thus, ALA can be used as an environmentally friendly herbicide that selectively kills weeds without causing damage to humans, animals and agricultural crops (see Rebeiz C. A. et al., Enzyme Microb. Technol., 6:390, 1984).
In addition to this use of ALA as herbicide, ALA has the effect of promoting plant growth by stimulating plant photosynthesis, inhibiting breathing and promoting carbon dioxide assimilation, when it is used at low concentrations (Hua Z., et al., Cancer Reasearch. 55:1723, 1995); Matsumoto T. H., et al., Weed Research, 37:60, 1992); Matsumoto T. H., et al., Pesticide Biochemistry, 48:214, 1994; Rebeiz N., et al., Photochem. Photobiol., 55:431, 1995). It was found that, when rice seeds were immersed in 1-3 ppm of an ALA solution for 1-48 hours and then sowed, the size and weight of the plant increased and the root growth of the plant was promoted.
In addition, ALA can also be used as an insecticide against noxious insects such as Trichopusia ni. In particular, regarding the insecticidal effect of ALA, the stage of action of ALA is very complicated, unlike conventional insecticides that are involved in a certain metabolic stage to exhibit their effect, and thus it is difficult for noxious insects to develop resistance to ALA, indicating that ALA can be used as an environmentally friendly insecticide. Furthermore, it was reported that ALA can be used as biologically active substances (such as skin cancer treating agents and antimicrobial drugs) in the medical field. Particularly, it was found that ALA can also be widely used as a photosensitizer in photodynamic therapy (PDP) for treating a variety of malignant tumors. Thus, studies on ALA have been actively conducted. Particularly, it was found that, when ALA was administered to a malignant tumor site, it rapidly increased the intracellular concentration of porphyrin, particularly protoporphyrin IX that is the final precursor in the heme biosynthesis pathway, and thus the tumor was damaged and killed by irradiation with visible light. However, it was found that, because normal cells absorb ALA at a slow rate and grow at a slow rate, compared to those of tumor cells, the concentration of protoporphyrin IX that is accumulated in normal cells is relatively low, and thus damage to normal cells by light irradiation is low.
Currently, ALA is produced using complicated organic synthesis processes (Beale S. I., et al., Phytochemistry, 18:441, 1979), but is not commercially profitable due to its high production cost. For this reason, studies have been conducted on ALA production methods based on fermentation of microorganisms, including Rhodobacter sphaeroides, Clostridium thermoaceticum, Methanobacterium thermoautotropicum, Agnemellum guadruplicum, Anacystis marina, and Chlorella vulgaris, and on the use thereof (Sasaki K., et al., J. Ferment. Technol., 65:511, 1987; Sasaki K., et al., Biotechnol. Lett., 15:859, 1993; Tanaka T., et al., Biotechnol. Lett., 13:589, 1991; Janschen R., et al., FEMS Microb. Lett., 12:167, 1981; Kipe-Not J. A. and Steven S. E., Plant Physiol., 65:126, 1980; Beale S. I. and Castelfranco P. A., Plant Physiol., 53:297, 1974).
It is known that ALA, a precursor of heme, is biosynthesized by two biosynthetic systems (C4 and C5 pathways). ALA by the C4 pathway, which is found in animals, fungi, bacteria, etc., is produced through the condensation of glycine and succinyl-CoA, and this condensation reaction is catalyzed by ALA synthase that is a pyridoxal phosphate-dependent enzyme. In addition, the C5 pathway is found in plants, algae, E. coli, etc.
The molecular biological ALA biosynthetic pathway was identified by isolation of ALA auxotrophs. It was found that ALA synthase genes in the C4 pathway are two isozymes (hemA and hemi), whereas the C5 pathway is composed of hemA, hemL and hemM genes.
In an attempt to increase the synthesis of ALA by microorganisms, both the supplement of precursors (glutamic acid, glycine and succinic acid) into culture media and studies on the isolation of lower fatty acids from organic waste resources and the effect of addition thereof were reported. In addition, studies on the increase in ALA production by pH and temperature control, oxygen supply, light irradiation for photosynthetic bacteria, etc., were also reported.
The present inventors have made extensive efforts to develop an efficient method capable of producing a large amount of 5-aminolevulinic acid, and as a result, have found that, when genes capable of producing 5-aminolevulinic acid are introduced into a microorganism that produces glutamic acid from the 5-carbon metabolic pathway among microbial metabolic pathways, the resulting microorganism is capable of producing 5-aminolevulinic acid in a high yield, thereby completing the present invention.